The lung is the critical site of entry for the most deadly form of anthrax infection, inhalation anthrax. Inhalation anthrax s relatively unique in that germinated vegetative bacteria of the etiologic agent of this disease, B.anthracis (Ba), do not cause disease at the site of entry. Instead Ba spores are taken up by resident lung cells and carried through lung lymphatic ducts to the thoracic lymph nodes (TLN), from which site Ba disseminates to cause the highly lethal, terminal phase of the disease. There remain many unanswered questions about this deadly disease. It is not known why vegetative Ba do not cause disease at the site of entry. It is not known what cells take the pathogen out of the lung to the TLN. Most importantly, it s not known what role Ba toxins (lethal toxin, LT; edema toxin, ET) play in this process. The overall goal of this proposal is to answer these questions. The current paradigm, based on mouse models and mouse cell lines, is that LT and ET do not play a significant role in the pathogenesis of inhalation anthrax. This may be correct for mice, but may not apply to humans. Mouse macrophage cell lines are very sensitive to the immunosuppressive effects of LT and express anthrax toxin receptors (ATR). In the last granting period we have determined that human alveolar macrophages (HAM) efficiently kill Ba vegetative bacteria, do not express ATR and are resistant to immunosuppressive effects of LT. We will test a new paradigm that holds that Ba toxins are very important in the early stages of inhalation anthrax and that the role of key lung cells is due to variable expression of the Ba toxins. We will test our new paradigm and answer the unanswered questions regarding inhalation anthrax in three Aims using a novel human lung organ culture model and a baboon inhalation model that is being developed by our colleague, Dr. Kurosawa. In the first Aim we will determine the human lung cells that internalize Ba and the state of the pathogen in these cells by exploiting our human lung organ culture model and by using flow cytometry, cell sorting, and quantitative confocal immunofluorescence microscopy. In the second Aim we will use a modification of the lung organ culture model, and tissue from Dr. Kurosawa's model to determine and confirm the cells that facilitate escape of Ba from the lung using techniques developed in the first Aim. In the final Aim, we will determine the role that Ba toxins play in inhalation anthrax, and whether downregulation of ATR in the lung organ culture model decreases internalization, survival and escape of the pathogen in human lung. This last set of experiments should provide a proof of concept to determine whether or not modulation of anthrax toxin receptors in human lung may be useful as preventative therapy for inhalation anthrax.